Several tree species belonging to the genus Populus are cultured mainly in the temperate regions of the northern hemisphere, because they grow quickly and find versatile use in applications such as afforestation trees and pulp wood. In general, these species are proliferated with asexual propagation using cuttings. Species belonging to the section Leuce which are applicable to forestation in Japan, as hard woods (broad leaf trees) for pulp use, are propagated by a root laying method because of their low rooting frequency by cuttings. However, since their large scale propagation by this method requires a prolonged period of time and a large nursery area, many studies have been made on their large scale propagation by a tissue culture to resolve these problems.
The tissue culture is a technique in which a part of plants is aseptically cultured under appropriate conditions to propagate and/or regenerate it into a mature plant. Since plants can be regenerated from a part of plant tissues within a marked short period of time by applying this technique, its applications to the large scale propagation of plantlets are expected. For example, there is a known process in which an anther or a stem of a species belonging to the section Leuce is cultured to induce growth of a callus as a dedifferentiated tissue from which plants are regenerated through differentiation of shoots (Propagation and Breeding of Arboreous Plant, Nogyo Tosho, pp. 248-256, 1989). However, since cali (calluses) are apt to cause genetic mutation during their growth, mutants are probably generated at the stage of callus growth even by using this method. This mutation becomes a problem when clone plantlets having the same genetic constitution are produced. Also, although this method has a shorter propagating period than the root laying method, the culturing must be completed through steps of inducing a callus from an explant such as an anther and a stem, growing the callus and differentiating shoots therefrom, and so several months are still required to cover the period from the beginning of the culturing of the explant to the completion of the differentiation of shoots. In consequence, it is also important to shorten this period when plantlets are produced in a large scale for industrial purpose, or selection and propagation of superior individuals are repeated for breeding. Application of the method to the creation of an alien gene-transformed hybrid aspen (Populus sieboldii.times.Populus grandidentata) has been reported (Matsunaga et al., Abstract of Papers, Second Tree Molecular Biology Symposium, pp. 72-77, 1992). However, because of the above-described reasons, this method still has practical problems such as low efficiency in obtaining clone plantlets having a desired gene introduced therein and prolonged period of time until completion of the regeneration of a plant.
In order to resolve these problems, an another tissue culture method is carried out in which plants are regenerated from a tissue thereof by differentiating adventitious buds without employing a step for inducing and growing a callus. The term "adventitious bud" as used herein means a bud induced by a certain means from a tissue which should not become a bud by nature, and a plant can be regenerated therefrom by culturing it under appropriate conditions similar to the differentiation of shoots from a callus. In this method, the differentiation of adventitious buds are induced under such conditions that a callus is not induced or allowed to grow if induced, so that the adventitious buds are differentiated from a shoot used as the material directly or from a slightly grown callus. In consequence, this method is relatively free from the problem of causing genetic mutation during the culturing step and requires only a shorter period of time for regenerating a plant than the redifferentiation method which always requires steps for the inducing and growing a callus and redifferentiating shoots from the callus. This method is, therefore, useful for the above-described industrial and breeding purposes and is now frequently used as a means for efficiently obtaining a transformed plant in which a gene is introduced by using an agrobacterium method.
In using a tissue culture technique, it is necessary to select a plant tissue to be cultured, a medium composition to be used in the culturing and other conditions such as temperature and light, depending on the purpose of the culturing. However, combinations of these conditions and the results obtained thereby do not always show a certain relationship, and it is rather common that the same result is not obtained when different species or even different varieties of the plant are used as the materials, respectively. For example, according to a report regarding detailed examination of the effects of plant hormone compositions in a culture medium and plant varieties (genotypes) on the differentiation of adventitious buds, the adventitious bud differentiation ratio varies within the range of from 0 to 100% depending on the difference in varieties even when the same basal medium supplemented with the same kind and amount of a plant hormone is used (Coleman et al., Plant Cell Reports, vol. 9, pp. 165-167, 1989). In consequence, the most important subject to be solved when a plant tissue culture technique is used is to find the optimum culture conditions depending on each plant to be used as the material and each purpose of the culturing. However, studies on their propagation by the tissue culture technique in the field of arboreous plants are slower in progress than those of herbaceous plants such as vegetables, flowers and ornamental plants. Accordingly, even when the adventitious bud differentiation is studied on arboreous plants, the current studies are limited to model studies in which the conditions used for the adventitious bud differentiation of the tobacco plant, which belongs to the herbaceous plant, are used as such merely changing the plant hormone composition in the medium, and a variety that can induce differentiation of adventitious buds relatively easily under such conditions is used as the material. For example, regeneration of a plant by adventitious bud differentiation from a leaf of a plant belonging to the genus Populus has been reported (Fillatti et al., Molecular General Genetics, vol. 206, pp. 192-199, 1987). However, a surprisingly limited amount of information exists on the studies of culture conditions including medium compositions which were conducted to induce efficient adventitious bud differentiation in practically useful arboreous plants.